Amides, adipic acid

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 17 March, 2020 to 29 april, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Version / remarks:

adopted 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

Test System: BALB/cMice
Scientific Name : Mus musculus
Justification: As this test system show good sensitization response to the positive control.
Source: Animal Breeding, RCC Laboratories India Private Limited, Genome Valley, Turkapally, Shameerpet (Manda!), Ranga Reddy District, Hyderabad - 500 078 India.

Pretest
Total number of Animals used: 8 Females (females were nulliparous and non-pregnant)

Main Study
Number of Animals per Group: 5 Females (females were nulliparous and non-pregnant)
Number of Groups: 6
Total Number of Animals: 30
Age when Treated: 11- 13 Weeks
Body weight when treated : Females: 21.72 to 24.52 g

Identification: By unique cage number and individual animal numbers marked with indelible marker pen on the tail. The animals were marked (towards the tip of tail) with red color for the temporary animal numbers at start of acclimatization. The animals were marked with black color for permanent animal numbers (towards the base of tail) before the start of test item administration.

Randomization: Animals were selected and grouped based on stratified randomization by using body weights. Computerized statistical analysis was used for randomization

Acclimatization: Under laboratory conditions for 07 days, after veterinary examination. Only animals without visible signs of illness were used for the study.

Environment
The animal room was air-conditioned with adequate (above 10) air changes per hour. The air was continuously monitored for temperature and relative humidity. The ranges for room temperature and relative humidity were 22.9 to 24.5°C and 50 to 65%, respectively. The animals were provided with a light cycle of 12 hours light and 12 hours dark.

Accomodation
Individually in Polycarbonate cages (approximate internal dimensions of 214 mm x 154 mm x 138 mm) with paddy husk bedding. Results of analyses for contaminants will be archived at RCC Laboratories India Private Limited.

Enrichment
Tunnels were provided for individually housed animals.

Diet
Teklad Certified Global 14% Protein Rodent Maintenance Diet (Lot Number: 2014C-091719MA) from ENVIGO was provided ad libitum. Results of analyses for contaminants will be archived at RCC Laboratories India Private Limited.

Water
UV purified water was provided ad libitum. Results of bacteriological, chemical and contaminant analyses will be archived at RCC Laboratories India Private Limited.

Vehicle:

dimethyl sulphoxide

Details on study design:

Dose formulation
The dose levels are in terms of the test substance as supplied. The dose formulations were prepared shortly before each dosing. The test substance was formulated in vehicle. The quantity of test substance was mixed with vehicle and the final volume was made up to 1000 µl for each group in a separate micro centrifuge tube. Homogeneity of the test substance in the vehicle was maintained during administration using a vortex mixer.

Note: DMSO (Dimethyl sulphoxide) was used as a vehicle for treatment group (Test substance) whereas acetone+ Olive Oil (4:1) were used as a vehicle for positive control group (Eugenol).

Selection of test substance concentrations for main study
A pretest was conducted to find out the appropriate test substance concentrations for main study. Two Female mice were used for each dose concentration. The dose concentrations used in the pretest are 10%, 25%, 50% and 100%. No mortality was observed at the concentrations of 10%, 25%, 50% and 100%. No clinical signs were observed at the concentrations of 10%, 25%, 50% and 100%. Animals were observed for body weight on Day 1 (prior to test substance application) and Day 6. Ears were observed for erythema on Day 1-6. Ear thickness was measured on Day 1 (pre dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Ear thickness and body weights were well within the normal range and no erythema was observed at the tested concentrations. The doses for the main study was selected based on the results of pretest and were described in an amendment to study plan. Pretest was performed before study initiation date and hence, excluded from statement of GLP. The doses selected for the main study was 10%, 25%, 50% and 100% for low, low mid, high mid and high doses, respectively. Considering the mortality, clinical signs and solubility of the test substance in the vehicle, the highest non-toxic concentration (100%) was selected for the main study.

Treatment
On Day 1, 2 and 3, the formulated test substance was applied to the dorsum of both the ears in a volume of 25 µL/ear using micropipette.

Justification: Dermal application is considered to be an appropriate application method as it is a possible route of human exposure during manufacture, handling and use of the test substance.

Dosing with BrdU
BrdU preparation
200 mg of BrdU was weighed into a tarred glass beaker on a weighing balance. 3 mL of phosphate buffer saline was added to beaker containing BrdU and mixed properly by using glass rod. This was then transferred to a 20 mL volumetric flask. Sufficient quantity of phosphate buffer saline was added to make up the required volume of solution (WN) to 20 mL. This was then transferred back to the beaker and mixed properly. The 10 mg/mL PBS solution containing BrdU was administered intraperitoneally to all the animals at a dose level of 5 mg/animal (0.5 mL/animal) on day 5. The BrdU solution was prepared freshly before administration.

Observation
MortalityNiability
Twice daily during the acclimatization period and during treatment and observation days, once on day of receipt and sacrifice.

Body Weights
On test days l (prior to test substance application) and 6 (Necropsy).

Clinical Signs
Once daily during the acclimatization period, at least twice daily approximately at 1st and 4th hours after test substance administration and on test day 1- 3, day 5 and once on day 4 and day 6.

Skin Reaction (Both Ears) Ear thickness
The skin reaction was assessed on days 1- 6 according to the numerical scoring system. Ear thickness was measured on Day I (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6.

Pathology
Necropsy
All the treated animals were sacrificed at the end of the observation period by carbon dioxide asphyxiation in euthanasia chamber. Gross/macroscopic pathological changes were observed and recorded. The auricular lymph nodes were excised from both ears of all the animals. The auricular lymph nodes were weighed and stored in ice-chilled Phosphate Buffer Saline (PBS) and kept in deep freezer (-20·C) until further processing. No organ and tissues were retained

6BrdU: ELISA
The incorporation of BrdU into lymph node cells was determined using a commercial cell proliferation assay kit (Roche Diagnostics). The assay procedure was as follows:

Lymph Node Cell (LNC) suspension Preparation
• The collected auricular lymph nodes were crushed using small pestle.
• 0.3 ml of PBS (Phosphate Buffer Saline) was added and the suspension was collected in a centrifuge tube.
• This suspension was pass through a nylon mesh (70µm). The volume of cell suspension was made up to 15 ml with PBS.

Assay Flow (BrdU- ELISA)
1. LNC suspension was centrifuged at 2000 rpm for ten minutes
2. ¾ th of supernatant solution was removed.
3. The cell suspension (100 µL) was added to Micro well plate (3 replicates /sample) and thoroughly mixed. This micro well plate was dried completely in a hot air oven at 45°C.
4. 200 µL of Fix-Denat solution was added to each well and allowed to stand for 30 minutes.
5. Fix-Denat solution was removed completely after 30 minutes.
6. 100 µL of anti-BrdU-POD antibody working solution was added and allowed to react for 1 hour.
7. Anti-BrdU-POD antibody working solution was removed completely after 1 hour.
8. 200 µL of wash solution (PBS) was added and washed the wells by pipetting 10 times. The solution was completely removed and discarded. Same step was repeated twice.
9. 100 µL of TMB (Tetra methyl Benzidine) substrate solution was added and allowed to stand for 15 minutes at room temperature in a dark place.
10. The absorbance was measured at 370 nm using spectramax reader.

Calculation
Stimulation Index (SI) was calculated as follows:
OD values for each test animal/ mean OD values for concurrent Negative control group

Accetability of the assay
The positive control response at an exposure level expected to give an increase in the SI greater than or equal to 1.6 over the negative control.
The SI >14 should be considered to be an excessive response. Induction at this level is considered to be not reproducible.
The SI 2:: 1.6 value of test substance is considered to be a positive response and the test substance a potential skin sensitizer.

Data compilation
Mortality/Viability, Clinical Signs, Skin Reactions, Ear Thickness measurements, Body Weights and Organ Weights were recorded on data sheets. All data generated by the 96-well plate ELISA reader was printed after reading and considered as raw data and excel sheet were used to calculate the Stimulation index. Data from individual animal was reported in tabular form. The Data was reported as mean± SD (standard deviation).

Positive control substance(s):

eugenol (CAS No 97-53-0)

Key result

Parameter:

SI

Value:

ca. 1.07

Test group / Remarks:

10% test substance

Key result

Parameter:

SI

Value:

ca. 1.111

Test group / Remarks:

25% test substance

Key result

Parameter:

SI

Value:

ca. 1.119

Test group / Remarks:

50% test substance

Key result

Parameter:

SI

Value:

ca. 3.954

Test group / Remarks:

Positive control

Key result

Parameter:

SI

Value:

ca. 1.154

Test group / Remarks:

100% test substance

Cellular proliferation data / Observations:

Mortality
No mortality was observed in any of the animals treated during experimental period.

Clinical signs
All the animals appeared normal throughout the acclimatization period. All the animals appeared normal throughout the study observation period.

Body weights
The body weight of the animals was within the normal range of variability commonly recorded for this species, strain and age.

Skin reaction
The test substance did not induce any degree of erythema on the both ears of all animals in control and treatment groups on Day 1 to Day 6. Animals in positive control group showed well defined erythema on both the ears on Day 1 to Day 6.

Ear thickness
Ear thickness measurements in treated group animals were comparable with negative control group. Whereas ear thickness was slightly increased in positive control group compared with negative control group.

Macroscopic findings
No abnormalities were observed externally and internally in negative control, low, low mid, high mid and high dose group animals at necropsy. Externally both ears (Bilateral) found reddened, internally the size of the auricular lymph nodes (Bilateral) was enlarged slightly in positive control group at terminal sacrifice.

Organ weight
The size of the auricular lymph nodes was enlarged slightly in positive control group compared to negative control group.

BrdU ELISA results
A BrdU ELISA measurement with cell suspension of Auricular lymph nodes resulted in mean stimulation indexes of 1.070, 1.111, 1.119 and 1.154 at concentrations of 10, 25, 50 and 100%, respectively. Stimulation Index (SI) value of positive control was 3.954 and thus confirmed the validity of the test system.

Conclusion
The Stimulation Index (SI) of animals treated with test substance at concentrations of 10, 25, 50 and 100% was less than 1.6. Hence, the test substance is not a potential skin sensitizer.

Results

Table 1: Mortality, viability and clinical signs

Group	Dose	Animal Number	Sex	Test day
				1		2		3		4	5		6
1	0%(NC)	01	Female	1	1	1	1	1	1	1	1	1	1
		02	Female	1	1	l	1	1	l	1	1	1	1
		03	Female	1	1	1	1	1	1	l	1	1	1
		04	Female	l	1	1	1	1	1	1	1	1	1
		05	Female	1	1	1	I	1	1	l	1	1	1
2	10%(L)	06	Female	1	1	1	1	1	1	1	1	1	1
		07	Female	1	1	I	1	1	1	1	1	1	1
		08	Female	1	1	1	1	1	1	I	1	1	l
		09	Female	1	1	1	1	1	l	1	1	1	1
		10	Female	1	1	I	I	1	1	1	1	I	1
3	25%(LM)	11	Female	1	1	1	1	1	1	1	1	1	1
		12	Female	1	1	1	1	1	l	1	1	1	1
		13	Female	1	l	1	1	1	1	1	1	1	1
		14	Female	1	1	1	1	1	1	1	1	1	1
		15	Female	1	1	1	1	1	1	1	1	1	1
4	50% (HM)	16	Female	l	1	1	1	1	1	1	1	1	1
		17	Female	1	1	1	1	1	1	l	1	1	l
		18	Female	1	1	1	1	1	1	1	1	1	1
		19	Female	1	1	1	1	1	l	1	1	l	1
		20	Female	1	1	1	1	1	1	1	1	1	1
5	100% (H)	21	Female	1	1	1	1	1	1	1	1	1	I
		22	Female	1	1	I	1	1	1	1	1	1	l
		23	Female	1	1	1	1	1	1	1	1	1	1
		24	Female	I	1	1	1	1	1	1	1	1	1
		25	Female	1	1	I	1	1	1	1	1	1	1
6	25%(PC)	26	Female	1	1	1	1	I	1	1	1	1	1
		27	Female	1	1	1	1	1	1	1	1	1	1
		28	Female	1	1	1	1	1	1	1	1	1	1
		29	Female	1	1	1	1	1	l	1	l	1	l
		30	Female	1	1	1	1	1	1	1	1	1	1

Key: %=percentage, l= Normal, NC-Negative Control, L-Low,   LM-Low Mid, HM- High Mid, H-High, PC-Positive Control

No clinical signs were evident in any animal during the acclimatization period.

Table 2: Body weight

Group	Dose	Animal Number	Sex	Test day 1 (pre-treatment) (g)	Test day 6 (g)
1	0% (NC)	01	Female	24.52	25.11
		02	Female	22.73	23.85
		03	Female	23.10	24.08
		04	Female	21.95	23.29
		05	Female	22.18	23.27
2	10% (L)	06	Female	23.78	25.75
		07	Female	22.69	24.41
		08	Female	22.50	23.41
		09	Female	21.83	23.70
		10	Female	22.13	23.89
3	25% (LM)	11	Female	23.62	24.70
		12	Female	22.75	23.43
		13	Female	22.43	23.25
		14	Female	21.76	23.84
		15	Female	21.91	23.33
4	50% (HM)	16	Female	23.29	23.96
		17	Female	22.80	24.37
		18	Female	22.69	24.33
		19	Female	22.15	23.52
		20	Female	21.90	22.84
5	100% (H)	21	Female	23.24	23.93
		22	Female	22.86	24.04
		23	Female	22.40	23.74
		24	Female	22.19	24.02
		25	Female	21.95	23.44
6	25% (PC)	26	Female	23.16	24.43
		27	Female	22.85	23.80
		28	Female	22.22	23.46
		29	Female	22.30	23.72
		30	Female	21.72	22.95

Key: %= percentage, g=gram, NC-Negative Control, L-Low, LM-Low Mid, HM- High Mid, H-High, PC-Positive Control

Table 3: Skin reactions

Reaction Scores
Group	Dose	Animal Number	Sex	Site ofApplication	Observation Day
					1	2	3	4	5	6
1	0% (NC)	01	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		02	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		03	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		04	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		05	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
2	10% (L)	06	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		07	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		08	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		09	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		10	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0

Key: NC-Negative Control, L-Low.

Reaction Scores
Group	Dose	AnimalNumber	Sex	Site ofApplication	Observation Day
					1	2	3	4	5	6
3	25% (LM)	11	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		12	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		13	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		14	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		15	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
4	50% (HM)	16	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		17	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		18	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		19	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		20	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0

Key: LM-Low Mid, HM-High Mid

Reaction Scores
Group	Dose	Animal Number	Sex	Site of Application	Observation Day
					1	2	3	4	5	6
5	100% (H)	21	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		22	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		23	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		24	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		25	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
6	25% (PC)	26	Female	Right Ear	0	2	2	2	2	2
				Left Ear	0	2	2	2	2	2
		27	Female	Right Ear	0	2	2	2	2	2
				Left Ear	0	2	2	2	2	2
		28	Female	Right Ear	0	2	2	2	2	2
				Left Ear	0	2	2	2	2	2
		29	Female	Right Ear	0	2	2	2	2	2
				Left Ear	0	2	2	2	2	2
		30	Female	Right Ear	0	2	2	2	2	2
				Left Ear	0	2	2	2	2	2

Key: H-High; PC-Positive Control

Table 4: Ear thickness on Day 1

Group	Dose	Animal Number	Sex	Site of Application	Ear Thickness (mm)	Mean	Mean±SD
1	0% (NC)	01	Female	Right Ear	0.280	0.279	0.280±0.0098
				Left Ear	0.278
		02	Female	Right Ear	0.297	0.296
				Left Ear	0.294
		03	Female	Right Ear	0.272	0.271
				Left Ear	0.269
		04	Female	Right Ear	0.274	0.273
				Left Ear	0.272
		05	Female	Right Ear	0.283	0.282
				Left Ear	0.281
2	10% (L)	06	Female	Right Ear	0.286	0.285	0.285±0.0061
				Left Ear	0.284
		07	Female	Right Ear	0.291	0.290
				Left Ear	0.289
		08	Female	Right Ear	0.295	0.294
				Left Ear	0.292
		09	Female	Right Ear	0.288	0.287
				Left Ear	0.286
		10	Female	Right Ear	0.302	0.301
				Left Ear	0.299

Key: mm-Millimeter; NC-Negative Control, L-Low

Group	Dose	AnimalNumber	Sex	Site of Aoolication	Ear Thickness (mm)	Mean	Mean±SD
3	25%(LM)	11	Female	Right Ear	0.294	0.293	0.300±0.0084
				Left Ear	0.291
		12	Female	Right Ear	0.308	0.307
				Left Ear	0.306
		13	Female	Right Ear	0.312	0.311
				Left Ear	0.309
		14	Female	Right Ear	0.292	0.291
				Left Ear	0.289
		15	Female	Right Ear	0.301	0.300
				Left Ear	0.298
4	50% (HM)	16	Female	Right Ear	0.298	0.297	0.298±0.0075
				Left Ear	0.295
		17	Female	Right Ear	0.309	0.308
				Left Ear	0.307
		18	Female	Right Ear	0.303	0.302
				Left Ear	0.301
		19	Female	Right Ear	0.289	0.288
				Left Ear	0.287
		20	Female	Right Ear	0.297	0.296
				Left Ear	0.294

Key: mm-Millimeter, LM-Low Mid, HM-High Mid

Group	Dose	AnimalNumber	Sex	Site ofApplication	Ear Thickness (mm)	Mean	Mean±SD
5	100% (H)	21	Female	Right Ear	0.314	0.313	0.304±0.0062
				Left Ear	0.311
		22	Female	Right Ear	0.299	0.298
				Left Ear	0.297
		23	Female	Right Ear	0.308	0.307
				Left Ear	0.305
		24	Female	Right Ear	0.299	0.298
				Left Ear	0.297
		25	Female	Right Ear	0.307	0.306
				Left Ear	0.304
6	25% (PC)	26	Female	Right Ear	0.294	0.293	0.299±0.0100
				Left Ear	0.292
		27	Female	Right Ear	0.315	0.314
				Left Ear	0.313
		28	Female	Right Ear	0.304	0.303
				Left Ear	0.301
		29	Female	Right Ear	0.289	0.288
				Left Ear	0.287
		30	Female	Right Ear	0.298	0.297
				Left Ear	0.296

Key: mm-Millimeter, H-High, PC-Positive Control

Table 5: Ear thickness on Day 3

Group	Dose	Animal Number	Sex	Site ofApplication	Ear Thickness (mm)	Mean	Mean±SD
I	0% (NC)	01	Female	Right Ear	0.282	0.281	0.282 ± 0.0095
				Left Ear	0.279
		02	Female	Right Ear	0.299	0.298
				Left Ear	0.296
		03	Female	Right Ear	0.275	0.274
				Left Ear	0.273
		04	Female	Right Ear	0.276	0.275
				Left Ear	0.274
		05	Female	Right Ear	0.286	0.285
				Left Ear	0.284
2	10% (L)	06	Female	Right Ear	0.288	0.287	0.287 ± 0.0061
				Left Ear	0.286
		07	Female	Right Ear	0.294	0.293
				Left Ear	0.292
		08	Female	Right Ear	0.297	0.296
				Left Ear	0.294
		09	Female	Right Ear	0.291	0.290
				Left Ear	0.289
		10	Female	Right Ear	0.304	0.303
				Left Ear	0.302

Key: mm-Millimeter; NC-Negative Control, L-Low

Group	Dose	AnimalNumber	Sex	Site of Aoolication	Ear Thickness (mm)	Mean	Mean±SD
3	25% (LM)	11	Female	Right Ear	0.296	0.295	0.303± 0.0081
				Left Ear	0.294
		12	Female	Right Ear	0.311	0.310
				Left Ear	0.308
		13	Female	Right Ear	0.314	0.313
				Left Ear	0.311
		14	Female	Right Ear	0.295	0.294
				Left Ear	0.292
		15	Female	Right Ear	0.304	0.303
				Left Ear	0.301
4	50% (HM)	16	Female	Right Ear	0.301	0.300	0.301± 0.0074
				Left Ear	0.298
		17	Female	Right Ear	0.312	0.311
				Left Ear	0.309
		18	Female	Right Ear	0.305	0.304
				Left Ear	0.303
		19	Female	Right Ear	0.291	0.291
				Left Ear	0.290
		20	Female	Right Ear	0.299	0.298
				Left Ear	0.297

Key: mm-Millimeter, LM-Low Mid, HM-High Mid

Group	Dose	Animal Number	Sex	Site of Application	Ear Thickness (mm)	Mean	Mean±SD
5	100% (H)	21	Female	Right Ear	0.317	0.316	0.307 ± 0.0064
				Left Ear	0.314
		22	Female	Right Ear	0.302	0.301
				Left Ear	0.300
		23	Female	Right Ear	0.311	0.310
				Left Ear	0.308
		24	Female	Right Ear	0.302	0.301
				Left Ear	0.299
		25	Female	Right Ear	0.311	0.310
				Left Ear	0.308
6	25% (PC)	26	Female	Right Ear	0.552	0.552	0.541± 0.0140
				Left Ear	0.551
		27	Female	Right Ear	0.528	0.526
				Left Ear	0.524
		28	Female	Right Ear	0.561	0.560
				Left Ear	0.558
		29	Female	Right Ear	0.537	0.536
				Left Ear	0.535
		30	Female	Right Ear	0.533	0.532
				Left Ear	0.531

Key: mm-Millimeter, H-High, PC-Positive Control

Table 6: Ear thickness on Day 6

Group	Dose	Animal Number	Sex	Site ofApplication	Ear Thickness (mm)	Mean	Mean±SD
1	0% (NC)	01	Female	Right Ear	0.284	0.283	0.285±0.0093
				Left Ear	0.282
		02	Female	Right Ear	0.300	0.300
				Left Ear	0.299
		03	Female	Right Ear	0.277	0.276
				Left Ear	0.275
		04	Female	Right Ear	0.279	0.278
				Left Ear	0.277
		05	Female	Right Ear	0.288	0.287
				Left Ear	0.286
2	10% (L)	06	Female	Right Ear	0.289	0.288	0.288±0.0064
				Left Ear	0.287
		07	Female	Right Ear	0.297	0.296
				Left Ear	0.295
		08	Female	Right Ear	0.300	0.299
				Left Ear	0.298
		09	Female	Right Ear	0.294	0.293
				Left Ear	0.292
		10	Female	Right Ear	0.306	0.305
				Left Ear	0.304

Key: mm-Millimeter; NC-Negative Control, L-Low

Group	Dose	Animal Number	Sex	Site ofApplication	Ear Thickness (mm)	Mean	Mean±SD
3	25% (LM)	11	Female	Right Ear	0.299	0.298	0.306 ± 0.0081
				Left Ear	0.297
		12	Female	Right Ear	0.314	0.313
				Left Ear	0.311
		13	Female	Right Ear	0.317	0.316
				Left Ear	0.314
		14	Female	Right Ear	0.298	0.297
				Left Ear	0.295
		15	Female	Right Ear	0.307	0.306
				Left Ear	0.304
4	50% (HM)	16	Female	Right Ear	0.304	0.303	0.303± 0.0075
				Left Ear	0.301
		17	Female	Right Ear	0.314	0.313
				Left Ear	0.312
		18	Female	Right Ear	0.308	0.307
				Left Ear	0.306
		19	Female	Right Ear	0.294	0.293
				Left Ear	0.292
		20	Female	Right Ear	0.302	0.301
				Left Ear	0.299

Key: mm-Millimeter, LM-Low Mid, HM-High Mid

Group	Dose	AnimalNumber	Sex	Site ofApplication	Ear Thickness (mm)	Mean	Mean±SD
5	100% (H)	21	Female	Right Ear	0.319	0.318	0.310±0.0062
				Left Ear	0.316
		22	Female	Right Ear	0.305	0.304
				Left Ear	0.302
		23	Female	Right Ear	0.314	0.313
				Left Ear	0.311
		24	Female	Right Ear	0.305	0.304
				Left Ear	0.302
		25	Female	Right Ear	0.314	0.313
				Left Ear	0.311
6	25% (PC)	26	Female	Right Ear	0.555	0.554	0.544±0.0140
				Left Ear	0.553
		27	Female	Right Ear	0.531	0.530
				Left Ear	0.528
		28	Female	Right Ear	0.564	0.563
				Left Ear	0.561
		29	Female	Right Ear	0.541	0.540
				Left Ear	0.539
		30	Female	Right Ear	0.535	0.534
				Left Ear	0.532

Key: mm-Millimeter, H-High, PC-Positive Control

Table 7: Macroscopic finding

Group	Dose	Animal Number	Sex	Mode of Death	Macroscopic Finding
					External	Internal
I	0% (NC)	01	Female	Terminal Sacrifice	NAD	NAD
		02	Female	Terminal Sacrifice	NAD	NAD
		03	Female	Terminal Sacrifice	NAD	NAD
		04	Female	Terminal Sacrifice	NAD	NAD
		05	Female	Terminal Sacrifice	NAD	NAD
2	10% (L)	06	Female	Terminal Sacrifice	NAD	NAD
		07	Female	Terminal Sacrifice	NAD	NAD
		08	Female	Terminal Sacrifice	NAD	NAD
		09	Female	Terminal Sacrifice	NAD	NAD
		10	Female	Terminal Sacrifice	NAD	NAD
3	25% (LM)	11	Female	Terminal Sacrifice	NAD	NAD
		12	Female	Terminal Sacrifice	NAD	NAD
		13	Female	Terminal Sacrifice	NAD	NAD
		14	Female	Terminal Sacrifice	NAD	NAD
		15	Female	Terminal Sacrifice	NAD	NAD
4	50% (HM)	16	Female	Terminal Sacrifice	NAD	NAD
		17	Female	Terminal Sacrifice	NAD	NAD
		18	Female	Terminal Sacrifice	NAD	NAD
		19	Female	Terminal Sacrifice	NAD	NAD
		20	Female	Terminal Sacrifice	NAD	NAD

Note:Dose expressed in concentration (%)

Key: NC-Negative control, L-Low, LM-Low Mid, HM-High Mid, NAD- No Abnormality Detected

Group	Dose	Animal Number	Sex	Mode of Death	Macroscopic Finding
					External	Internal
5	100% (H)	21	Female	Terminal Sacrifice	NAD	NAD
		22	Female	Terminal Sacrifice	NAD	NAD
		23	Female	Terminal Sacrifice	NAD	NAD
		24	Female	Terminal Sacrifice	NAD	NAD
		25	Female	Terminal Sacrifice	NAD	NAD
6	25% (PC)	26	Female	Terminal Sacrifice	Ears (Bilateral)- Reddened, slightly	Auricular lymph node (Bilateral)- enlarged,slightly
		27	Female	Terminal Sacrifice	Ears (Bilateral)- Reddened, slightly	Auricularlymph node (Bilateral) - enlarged,slightly
		28	Female	Terminal Sacrifice	Ears (Bilateral)- Reddened, slightly	Auricularlymph node (Bilateral)- enlarged,slightly
		29	Female	Terminal Sacrifice	Ears (Bilateral)- Reddened, slightly	Auricularlymphnode (Bilateral) - enlarged,slightly
		30	Female	Terminal Sacrifice	Ears (Bilateral)- Reddened, slightly	Auricularlymph node (Bilateral) - enlarged,slightly

Note:Dose expressed in concentration (%)

Key: H- High, PC- Positive Control, NAD- No Abnormality Detected

Table 8: Organ weight

Group	Dose	Animal Number	Sex	Auricular Lymph nodes (gram)
1	0% (NC)	01	Female	0.0069
		02	Female	0.0072
		03	Female	0.0064
		04	Female	0.0069
		05	Female	0.0054
2	10% (L)	06	Female	0.0061
		07	Female	0.0065
		08	Female	0.0076
		09	Female	0.0068
		10	Female	0.0059
3	25% (LM)	11	Female	0.0074
		12	Female	0.0063
		13	Female	0.0071
		14	Female	0.0068
		15	Female	0.0057
4	50% (HM)	16	Female	0.0059
		17	Female	0.0066
		18	Female	0.0069
		19	Female	0.0079
		20	Female	0.0082
5	100% (H)	21	Female	0.0068
		22	Female	0.0071
		23	Female	0.0083
		24	Female	0.0072
		25	Female	0.0069
6	25% (PC)	26	Female	0.0137
		27	Female	0.0124
		28	Female	0.0129
		29	Female	0.0141
		30	Female	0.0133

Key: NC-Negative control, L-Low, LM-Low Mid, HM- High Mid, H- High, PC- Positive Control

Table 9: BrDU ELISA reading

Group	Dose	Animal Number	Sex	OD Readings
1	0% (NC)	01	Female	0.242
				0.234
				0.224
		02	Female	0.232
				0.238
				0.245
		03	Female	0.206
				0.199
				0.201
		04	Female	0.198
				0.207
				0.210
		05	Female	0.188
				0.179
				0.191
2	10% (L)	06	Female	0.236
				0.229
				0.245
		07	Female	0.228
				0.224
				0.233
		08	Female	0.258
				0.241
				0.245
		09	Female	0.217
				0.208
				0.203
		10	Female	0.216
				0.211
				0.224
3	25% (LM)	11	Female	0.261
				0.253
				0.254
		12	Female	0.247
				0.236
				0.231
		13	Female	0.228
				0.232
				0.234
		14	Female	0.237
				0.243
				0.241
		15	Female	0.223
				0.214
				0.217

Key: NC-Negative Control, L- Low, Low Mid

Group	Dose	Animal Number	Sex	OD Readings
4	50% (HM)	16	Female	0.242
				0.236
				0.229
		17	Female	0.224
				0.219
				0.231
		18	Female	0.252
				0.259
				0.264
		19	Female	0.233
				0.226
				0.239
		20	Female	0.229
				0.248
				0.241
5	100% (H)	21	Female	0.261
				0.269
				0.254
		22	Female	0.258
				0.243
				0.247
		23	Female	0.227
				0.234
				0.236
		24	Female	0.243
				0.239
				0.248
		25	Female	0.251
				0.242
				0.239

Key: HM- High Mid, H-High

Group	Dose	Animal Number	Sex	OD Readings
6	25% (PC)	26	Female	0.860
				0.883
				0.878
		27	Female	0.869
				0.843
				0.830
		28	Female	0.962
				0.953
				0.909
		29	Female	0.767
				0.754
				0.757
		30	Female	0.794
				0.797
				0.778

Key: PC-Positive Control

Table 10: LLNA-BrDU ELISA mean readings

Animal No.	Mean OD value	AnimalNo.	Mean OD value	AnimalNo.	Mean OD value	AnimalNo.	Mean OD value
Gl		G2		G3		G4
01	0.233	06	0.237	11	0.256	16	0.236
02	0.238	07	0.228	12	0.238	17	0.225
03	0.202	08	0.248	13	0.231	18	0.258
04	0.205	09	0.209	14	0.240	19	0.233
05	0.186	10	0.217	15	0.218	20	0.239

Key: OD=OpticalDensity

 

Animal No.	Mean OD value	Animal No.	Mean OD value
GS		G6
21	0.261	26	0.874
22	0.249	27	0.847
23	0.232	28	0.941
24	0.243	29	0.759
25	0.244	30	0.790

Key: OD=OpticalDensity

GI-Negative Control, G2-Low, G3-Low Mid, G4-High Mid, GS-High, G6-Positive Control

Table 11: Stimulation index

Group	Animal No.	SI value	Mean±SD	Minimum	Maximum
G2	06	1.113	1.070 ± 0.0729	0.981	1.164
	07	1.071
	08	1.164
	09	0.981
	10	1.019
G3	11	1.202	1.111 ± 0.0650	1.023	1.202
	12	1.117
	13	1.085
	14	1.127
	15	1.023
G4	16	1.108	1.119 ± 0.0575	1.056	1.211
	17	1.056
	18	1.211
	19	1.094
	20	1.122
GS	21	1.225	1.154 ± 0.0494	1.089	1.225
	22	1.169
	23	1.089
	24	1.141
	25	1.146
G6	26	4.103	3.955 ± 0.3357	3.563	4.418
	27	3.977
	28	4.418
	29	3.563
	30	3.709

Key: G2-Low, G3-Low Mid, G4-High Mid, GS-High, G6-Positive Control

Table 12: Pre-test

(1) Ear thickness on Day 1

Group	Dose	Animal Number	Sex	Site of Aoolication	Ear Thickness (mm)	Mean	Mean±SD
1	10%	01	Female	Right Ear	0.298	0.298	0.301± 0.0053
				Left Ear	0.297
		02		Right Ear	0.306	0.305
				Left Ear	0.304
2	25%	03	Female	Right Ear	0.319	0.318	0.313± 0.0067
				Left Ear	0.316
		04		Right Ear	0.309	0.308
				Left Ear	0.307
3	50%	05	Female	Right Ear	0.305	0.306	0.313± 0.0095
				Left Ear	0.307
		06		Right Ear	0.321	0.320
				Left Ear	0.318
4	100%	07	Female	Right Ear	0.299	0.299	0.300 ±.0021
				Left Ear	0.298
		08		Right Ear	0.302	0.302
				Left Ear	0.301

Key: mm-Millimeter

(2) Ear thickness on Day 3

Group	Dose	Animal Number	Sex	Site ofApplication	Ear Thickness (mm)	Mean	Mean±SD
1	10%	01	Female	Right Ear	0.300	0.300	0.303 ± 0.0046
				Left Ear	0.299
		02		Right Ear	0.307	0.306
				Left Ear	0.305
2	25%	03	Female	Right Ear	0.321	0.320	0.315 ± 0.0071
				Left Ear	0.318
		04		Right Ear	0.311	0.310
				Left Ear	0.308
3	50%	05	Female	Right Ear	0.308	0.309	0.316 ± 0.0092
				Left Ear	0.310
		06		Right Ear	0.323	0.322
				Left Ear	0.321
4	100%	07	Female	Right Ear	0.302	0.302	0.303 ± 0.0014
				Left Ear	0.301
		08		Right Ear	0.304	0.304
				Left Ear	0.303

Key: mm-Millimeter

(3) Ear thickness on Day 6

Group	Dose	Animal Number	Sex	Site ofApplication	Ear Thickness (mm)	Mean	Mean±SD
l	10%	01	Female	Right Ear	0.303	0.302	0.305 ± 0.0046
				Left Ear	0.300
		02		Right Ear	0.309	0.308
				Left Ear	0.307
2	25%	03	Female	Right Ear	0.323	0.322	0.317± 0.0067
				Left Ear	0.321
		04		Right Ear	0.314	0.313
				Left Ear	0.311
3	50%	05	Female	Right Ear	0.312	0.312	0.318±0.0014
				Left Ear	0.311
		06		Right Ear	0.326	0.325
				Left Ear	0.324
4	100%	07	Female	Right Ear	0.305	0.304	0.305 ± 0.0014
				Left Ear	0.303
		08		Right Ear	0.307	0.306
				Left Ear	0.305

Key: mm-Millimeter

Table 14: Body weight

Group	Dose	Animal Number	Sex	Test Day 1 (pre-treatment) (g)	Test Day 6(Necropsy)(g)
1	10%	01	Female	20.74	22.28
		02	Female	21.16	23.18
2	25%	03	Female	20.10	21.32
		04	Female	20.59	21.75
3	50%	05	Female	19.95	21.42
		06	Female	21.23	22.85
4	100%	07	Female	20.16	21.92
		08	Female	20.64	22.53

Note: g-gram

Table 15: Clinical signs

Group	Dose	Animal Number	Sex	Test day
				1		2		3		4	5	6
I	10%	01	Female	1	1	1	1	1	1	1	1	1
		02	Female	l	1	I	1	1	1	1	1	1
2	25%	03	Female	1	1	1	1	I	l	1	1	1
		04	Female	1	1	1	1	1	1	1	1	1
3	50%	05	Female	1	1	l	1	1	1	1	1	1
		06	Female	1	1	1	1	1	1	1	1	1
4	100%	07	Female	1	l	1	1	1	1	1	1	1
		08	Female	1	1	1	1	1	1	1	1	1

Key: l= Normal

Table 16: Dose foprmulation (pre-test)

Group	Name	Test Substance/Reference Substance Quantity	Vehicle (DMSO)	Final Concentration (%)
1	Low dose	100mg	1000 µI	10
2	Low mid	250mg	1000 µl	25
3	High mid	500mg	1000 µI	50
4	High dose	1000 mg	1000 µI	100

Note: DMSO- Dimethyl Sulphoxide

Table 17: Irritation scoring

Group	Dose	AnimalNumber	Sex	Site ofApplication	Observation day
					1	2	3	4	5	6
1	10%	01	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		02		Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
2	25%	03	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		04		Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
3	50%	05	Female	Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
		06		Right Ear	0	0	0	0	0	0
				Left Ear	0	0	0	0	0	0
4	100%	07	Female	Right Ear Left Ear	0	0	0	0	0	0
					0	0	0	0	0	0
		08		Right Ear Left Ear	0	0	0	0	0	0
					0	0	0	0	0	0

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance was not skin sensitizing.

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance according to OECD Guideline 442B (Local Lymph Node Assay: BrdU -ELISA method), in compliance with GLP. Healthy animals were allocated to six groups, each consisting of five female BALB/c mice. Animals in Group 1 (GI) were applied with vehicle (DMSO) and served as negative control (NC). The test substance was applied to both ears at the concentrations of 10% (low), 25% (low mid), 50% (high mid) and 100% (high) for dose groups G2, G3, G4 and G5, respectively. In addition, animals in Group 6 (positive control) received the reference substance eugenol (25%) in acetone: olive oil (4:1). The dorsum of both ears was used as treatment site. The animals were observed for mortality at least twice daily during the acclimatization, treatment and observation days and at least once on day of receipt and sacrifice. Body weights were recorded on test Day 1 (prior to test substance administration) and Day 6. The animals were observed for clinical signs once daily during the acclimatization period, twice daily i.e., approximately at 1 and 4 h after test substance administration on test Days 1, 2, 3, 5 and once on Days 4 and 6. The skin reactions (both ears) and ear thickness were measured on Days 1 to 6 and Days 1, 3 and 6, respectively. All animals were necropsied and examined macroscopically at the end of the observation period. The auricular lymph nodes were collected from both ears and analyzed with BrdU ELISA. All animals appeared normal throughout the acclimatization and experimental periods. Bodyweight was within the normal range of variability commonly recorded for this species, strain and age. No erythema was observed prior to the administration period. The vehicle did not cause any erythema in the negative control group. The test substance also did not induce any erythema in the low, low mid, high mid and high dose groups from Days 1 to 6. In the positive control group, all the animals showed well defined erythema on both the ears from Days 2 to 6. Ear thickness in the treated group animals was comparable with that of the negative control group, whereas ear thickness was slightly increased in the positive control group. At necropsy, no abnormalities were observed externally and internally in the negative control, low, low mid, high mid and high dose group animals, whereas externally ears (bilateral) were found to be reddened slightly and internally the size of the auricular lymph nodes (bilateral) was slightly increased in the positive control group in gross pathology. BrdU ELISA measurement with the positive control cell suspension of auricular lymph nodes resulted in a Stimulation Index (SI) value of 3.954. As the SI was greater than 1.6, sensitivity and validity of the test system were confirmed. BrdU ELISA measurement with treated groups (G2 to G5) resulted in SI of 1.070 - 1.154. As SI were less than 1.6, the test substance was considered to have no sensitization potential in the test system. Under the study conditions, the test substance was not sensitising to mouse skin (Madhu, 2020).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A study was conducted to determine the skin sensitisation potential of the test substance according to OECD Guideline 442B (Local Lymph Node Assay: BrdU -ELISA method), in compliance with GLP. Healthy animals were allocated to six groups, each consisting of five female BALB/c mice. Animals in Group 1 (GI) were applied with vehicle (DMSO) and served as negative control (NC). The test substance was applied to both ears at the concentrations of 10% (low), 25% (low mid), 50% (high mid) and 100% (high) for dose groups G2, G3, G4 and G5, respectively. In addition, animals in Group 6 (positive control) received the reference substance eugenol (25%) in acetone: olive oil (4:1). The dorsum of both ears was used as treatment site. The animals were observed for mortality at least twice daily during the acclimatization, treatment and observation days and at least once on day of receipt and sacrifice. Body weights were recorded on test Day 1 (prior to test substance administration) and Day 6. The animals were observed for clinical signs once daily during the acclimatization period, twice daily i.e., approximately at 1 and 4 h after test substance administration on test Days 1, 2, 3, 5 and once on Days 4 and 6. The skin reactions (both ears) and ear thickness were measured on Days 1 to 6 and Days 1, 3 and 6, respectively. All animals were necropsied and examined macroscopically at the end of the observation period. The auricular lymph nodes were collected from both ears and analyzed with BrdU ELISA. All animals appeared normal throughout the acclimatization and experimental periods. Bodyweight was within the normal range of variability commonly recorded for this species, strain and age. No erythema was observed prior to the administration period. The vehicle did not cause any erythema in the negative control group. The test substance also did not induce any erythema in the low, low mid, high mid and high dose groups from Days 1 to 6. In the positive control group, all the animals showed well defined erythema on both the ears from Days 2 to 6. Ear thickness in the treated group animals was comparable with that of the negative control group, whereas ear thickness was slightly increased in the positive control group. At necropsy, no abnormalities were observed externally and internally in the negative control, low, low mid, high mid and high dose group animals, whereas externally ears (bilateral) were found to be reddened slightly and internally the size of the auricular lymph nodes (bilateral) was slightly increased in the positive control group in gross pathology. BrdU ELISA measurement with the positive control cell suspension of auricular lymph nodes resulted in a Stimulation Index (SI) value of 3.954. As the SI was greater than 1.6, sensitivity and validity of the test system were confirmed. BrdU ELISA measurement with treated groups (G2 to G5) resulted in SI of 1.070 - 1.154. As SI were less than 1.6, the test substance was considered to have no sensitization potential in the test system. Under the study conditions, the test substance was not sensitising to mouse skin (Madhu, 2020).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of an in vivo LLNA study, the substance does not warrant classification for skin sensitisation according to EU CLP (Regulation EC/1272/2008) criteria.